PROJECT SUMMARY Human pluripotent stem cells represent a potentially unlimited source of cardiomyocytes (CMs) for the study of cardiovascular development and disease, as well as for use in regenerative therapies. Tissue engineering enables the generation of three-dimensional constructs that mimic native cardiac structure and function and serve as substrates for therapeutic development and biological interrogation. However, during native cardiac development, cardiomyocytes interact directly with other cell types such as epicardial cells (EpiCs) and cardiac fibroblasts (CFs) to achieve physiologic function. In order for engineered cardiac tissues to fully recapitulate native myocardium, they require the addition of cardiac non-myocyte cells. Although it is well appreciated that these non-myocyte cells contribute to stable tissue formation and can modulate cardiac function, the specific role of epicardial cells and epicardial-derived cardiac fibroblasts on cardiac microtissue function remains unknown. Thus, our primary objective of this project is to determine how bi-directional influences of epicardial- derived CFs and CMs affect engineered cardiac microtissue function and organization. Our central hypothesis is that heterotypic interactions between epicardial-derived CFs and CMs are critical to promote phenotypic and functional maturation of engineered cardiac tissues. This hypothesis is based on our ability to engineer cardiac microtissues of controlled compositions of CMs, EpiCs, or EpiC-CF. Specifically, in Aim 1 we will evaluate the phenotype of CFs that have been derived from human pluripotent stem cells through an epicardial intermediary step and determine their similarity to native CFs. In Aim 2 we will evaluate the heterotypic effects of epicardial- derived cardiac fibroblasts on engineered cardiac tissue function. In Aim 3, we will determine if heterotypic interactions with CMs induce epithelial-to-mesenchymal transition of epicardial cells, enabling cardiac fibroblast invasion of engineered cardiac constructs. Successful completion of this project will give insight into the specific roles epicardial cells and epicardial-derived fibroblasts have in promoting cardiac physiologic function. Armed with this knowledge, more accurate in vitro models of cardiac tissue can be achieved, enabling the study of human cardiac diseases.